66 M. Priest and F. Bezanilla
Importantly, conformational changes measured by fluorometry can be wholly
induced by small molecules and are in no way limited to changes in membrane
potential. In the ether-a-go-go potassium channel, for example, two conformational
changes of the S4 are observable: one that correlates with the movement of the
majority of the charge, and another that matches the magnesium-dependent, rate-
limiting step that precedes charge movement; altering magnesium levels shifts both
current and fluorescence recordings as expected (Bannister et al. 2005 ; Schönherr
et al. 2002 ).
3.4 Functional Site-Directed Fluorometry in Ligand-Gated Ion
Channels
Site-directed fluorometry can also be applied to ligand-gated ion channels. The
most frequently studied have been members of the pentameric, Cys-loop receptor
family, especially the GABA- and glycine-gated ion channels. In both it has been
found that probes placed in Loop E or the extracellular side of M2 typically follow
the dose-response curve of the receptor’s endogenous ligand, while those placed
elsewhere, such as loop F, do not (Chang and Weiss 2002 ; Khatri et al. 2009 ; Muroi
et al. 2006 ; Pless and Lynch 2009a; Pless et al. 2007 ; Zhang et al. 2009 ). Interest-
ingly, fluorescence changes from similar sites in the related nAChR did not follow
the ligand dose-response curve, but instead preceded it as changes in fluorescence
were seen at concentrations too low to activate the channel (Dahan et al. 2004 ;
Mourot et al. 2008 ). Fluorescence changes have also been observed that follow the
time course of desensitization from the extracellular side of the nAChR M2 (Dahan
et al. 2004 ) and from the interface between the transmembrane domain M1 and the
ligand binding domain in a glycine receptor (Wang and Lynch 2011 ).
Fluorometry has also uncovered interesting information regarding the effects of
different ligands on ion channel conformational changes. In Cys-loop receptors,
application of different full agonists produced very similar conformational changes
as determined by changes in fluorescence (Chang and Weiss 2002 ; Khatri et al.
2009 ; Mourot et al. 2008 ). However, application of irreversible agonists (Pless et al.
2007 ) or partial agonists (Dahan et al. 2004 ; Khatri et al. 2009 ; Pless and Lynch
2009b; Pless et al. 2007 ) produced fluorescence changes that were different at many
sites from those produced by full agonists and by each other. Similarly, competitive
antagonists induced fluorescence changes at some sites that were different from
baseline, suggesting that they produced a conformational change rather than simply
preventing an agonist from binding; furthermore, different competitive antagonists
produced different fluorescent patterns (Chang and Weiss 2002 ; Pless and Lynch
2009a; Zhang et al. 2009 ). Noncompetitive antagonists tended to induce fewer fluo-
rescence changes, likely due to having primarily internal effects that are difficult
to record through site-directed fluorometry (Chang and Weiss 2002 ; Mourot et al.
2008 ; Muroi et al. 2006 ; Pless et al. 2007 ; Zhang et al. 2009 ).